Regulation Of Cytochrome P450 2E1 Research Articles

Antioxidant effect of Terminalia arjuna extract against acetaminophen-induced hepatotoxicity via the regulation of cytochrome P450 2E1, phosphatidylinositol-3-kinase/protein kinase B

Aim: The present study explored the therapeutic in detail antioxidants and effect of aqueous Terminalia arjuna (TA) bark extract against acetaminophen (APAP) induced hepatotoxicity through studies on serum marker enzymes, phosphatidylinositol3kinase/protein kinase B (PI3K/AKT) pathway, CYP2E1 evaluations. Biochemical, antioxidant, cytochrome P450 2E1 (CYP2E1) enzyme, and PI3K/AKT cell signal enzymes were observed with the appropriate methods of study. Materials and Methods: The animals were divided into five groups (each having six animals): control group, Acetaminophen (APAP) toxic group, N-acetylcysteine (NAC) group, TA 250 mg/kg group, and TA 500 mg/kg group. APAP toxic dose of 750 mg/kg body weight was administered along with 0.5% of hydroxypropyl cellulose (vehicle) 24 h before sacrificing the animal. Results: The biochemical, antioxidant, Histopathological, CYP2E1 enzyme, PI3K, AKT protein expression analysis were shown increased antioxidant level, increased PI3K/AKT level, decreased liver function marker level and decreased CYP2E1 level in TA500 mg group compared with APAP toxic group (P < 0.01). The findings suggest that TA (500mg/kg) drug reduced Acetaminophen toxicity via antioxidant and molecular mechanisms. Conclusion: The present study concluded that TA 500 mg/kg high dose is more effective to restore the liver tissue through APAPinduced hepatotoxicity in Wistar albino rats.

A systematic evaluation of microRNAs in regulating human hepatic CYP2E1

Cytochrome P450 2E1 (CYP2E1) is an important drug metabolizing enzyme for processing numerous xenobiotics in the liver, including acetaminophen and ethanol. Previous studies have shown that microRNAs (miRNAs) can suppress CYP2E1 expression by binding to the 3′-untranslated region (3′-UTR) of its transcript. However, a systematic analysis of CYP2E1 regulation by miRNAs has not been described. Here, we applied in silico, in vivo, and in vitro approaches to investigate miRNAs involved in the regulation of CYP2E1. Initially, potential miRNA binding sites in the CYP2E1 mRNA transcript were identified and screened using in silico methods. Next, inverse correlations were found in human liver samples between the expression of CYP2E1 mRNA and the levels of two miRNA species, hsa-miR-214-3p and hsa-miR-942-5p. In a HepG2-derived CYP2E1 over-expression cell model, hsa-miR-214-3p exhibited strong suppression of CYP2E1 expression by targeting the coding region of its mRNA transcript, but hsa-miR-942-5p did not inhibit CYP2E1 levels. Electrophoretic mobility shift assays confirmed that hsa-miR-214-3p recruited other cellular protein factors to form stable complexes with specific sequences present in the CYP2E1 mRNA open reading frame. Transfection of HepaRG cells with hsa-miR-214-3p mimics inhibited expression of the endogenous CYP2E1 gene. Further, hsa-miR-214-3p mimics partially blocked ethanol-dependent increases in CYP2E1 mRNA and protein levels in HepG2 cells and they reduced the release of alanine aminotransferase from CYP2E1-overexpressing HepG2 cells exposed to acetaminophen. These results substantiate the suppressing effect of hsa-miR-214-3p on CYP2E1 expression.

Effect of BI-1 on insulin resistance through regulation of CYP2E1.

Diet-induced obesity is a major contributing factor to the progression of hepatic insulin resistance. Increased free fatty acids in liver enhances endoplasmic reticulum (ER) stress and production of reactive oxygen species (ROS), both are directly responsible for dysregulation of hepatic insulin signaling. BI-1, a recently studied ER stress regulator, was examined to investigate its association with ER stress and ROS in insulin resistance models. To induce obesity and insulin resistance, BI-1 wild type and BI-1 knock-out mice were fed a high-fat diet for 8 weeks. The BI-1 knock-out mice had hyperglycemia, was associated with impaired glucose and insulin tolerance under high-fat diet conditions. Increased activity of NADPH-dependent CYP reductase-associated cytochrome p450 2E1 (CYP2E1) and exacerbation of ER stress in the livers of BI-1 knock-out mice was also observed. Conversely, stable expression of BI-1 in HepG2 hepatocytes was shown to reduce palmitate-induced ER stress and CYP2E1-dependent ROS production, resulting in the preservation of intact insulin signaling. Stable expression of CYP2E1 led to increased ROS production and dysregulation of insulin signaling in hepatic cells, mimicking palmitate-mediated hepatic insulin resistance. We propose that BI-1 protects against obesity-induced hepatic insulin resistance by regulating CYP2E1 activity and ROS production.

RORα switches transcriptional mode of ERRγ that results in transcriptional repression of CYP2E1 under ethanol-exposure.

Increased cytochrome P450 2E1 (CYP2E1) expression is the main cause of oxidative stress, which exacerbates alcoholic liver diseases (ALDs). Estrogen-related receptor gamma (ERRγ) induces CYP2E1 expression and contributes to enhancing alcohol-induced liver injury. Retinoic acid-related orphan receptor alpha (RORα) has antioxidative functions; however, potential cross-talk between ERRγ and RORα in the regulation of CYP2E1 has not been studied. We report that RORα suppressed ERRγ-mediated CYP2E1 expression. A physical interaction of RORα with ERRγ at the ERRγ−response element in the CYP2E1 promoter was critical in this suppression. At this site, coregulator recruitment of ERRγ was switched from coactivator p300 to the nuclear receptor corepressor 1 in the presence of RORα. Cross-talk between ERRγ and RORα was demonstrated in vivo, in that administration of JC1–40, a RORα activator, significantly decreased both CYP2E1 expression and the signs of liver injury in ethanol-fed mice, and this was accompanied by coregulator switching. Thus, this non-classical RORα pathway switched the transcriptional mode of ERRγ, leading to repression of alcohol-induced CYP2E1 expression, and this finding may provide a new therapeutic strategy against ALDs.

Single-Nucleotide Polymorphisms in Cytochrome P450 2E1 (CYP2E1) 3'-Untranslated Region Affect the Regulation of CYP2E1 by miR-570.

Human cytochrome P450 2E1 (CYP2E1) catalyzes the metabolism of numerous xenobiotics, including acetaminophen and ethanol. CYP2E1 expression is known to be extensively regulated by post-transcriptional and post-translational mechanisms. A previous study had reported that a single-nucleotide polymorphism (SNP) 1561A>G in the 3'-untranslated region (3'-UTR) of CYP2E1 leads to a decreased CYP2E1 mRNA level in human peripheral blood mononuclear cells. In this study, we examined the possibility that microRNA(s) (miR) may be involved in the SNP-mediated modulation of CYP2E1 expression. Genotyping and sequencing analyses revealed that another SNP, 1556T>A, in the 3'-UTR was in complete linkage disequilibrium with the SNP 1561A>G. We termed the alleles with 1556T and 1561A or 1556A and 1561G haplotype I or II, respectively. A luciferase assay revealed that miR-570 recognizes the CYP2E1 3'-UTR of haplotype I but not haplotype II. Human embryonic kidney 293 (HEK293) cell lines stably expressing human CYP2E1 that included the 3'-UTR of haplotype I or II (HEK/2E1(I) or HEK/2E1(II) cells, respectively) were established. Overexpression of miR-570 significantly decreased the CYP2E1 protein level in the HEK/2E1(I) cells but not in the HEK/2E1(II) cells. In seven human livers with diplotype I/I, the CYP2E1 protein levels were inversely correlated with the miR-570 levels, but no relationship was observed in 25 human livers with diplotypes I/II and II/II. Collectively, it was demonstrated that human CYP2E1 was regulated by miR-570 in a genotype-dependent manner. This report describes the first proof that SNP in 3'-UTR of human P450 affects binding of miRNA to modulate the expression in the liver.

Estrogen-related receptor γ controls hepatic CB1 receptor-mediated CYP2E1 expression and oxidative liver injury by alcohol

BackgroundThe hepatic endocannabinoid system and cytochrome P450 2E1 (CYP2E1), a key enzyme causing alcohol-induced reactive oxygen species (ROS) generation, are major contributors to the pathogenesis of alcoholic liver disease. The...

Cytochrome P450 2E1 and hyperglycemia-induced liver injury

Cytochrome P450 2E1 (CYP2E1), a microsomal enzyme involved in xenobiotic metabolism and generation of oxidative stress, has been implicated in promoting liver injury. The review deals with the changes in various cellular pathways in liver linked with the changes in regulation of CYP2E1 under hyperglycemic conditions. Some of the hepatic abnormalities associated with hyperglycemia-mediated induction of CYP2E1 include increased oxidative stress, changes in mitochondrial structure and function, apoptosis, nitrosative stress, and increased ketone body accumulation. Thus, changes in regulation of CYP2E1 are associated with the injurious effects of hyperglycemia in liver.

Bax inhibitor 1 regulates ER-stress-induced ROS accumulation through the regulation of cytochrome P450 2E1

This study investigated the molecular mechanism by which Bax inhibitor 1 (BI1) abrogates the accumulation of reactive oxygen species (ROS) in the endoplasmic reticulum (ER). Electron uncoupling between NADPH-dependent cytochrome P450 reductase (NPR) and cytochrome P450 2E1 (P450 2E1) is a major source of ROS on the ER membrane. ER stress produced ROS accumulation and lipid peroxidation of the ER membrane, but BI1 reduced this accumulation. Under ER stress, expression of P450 2E1 in control cells was upregulated more than in BI1-overexpressing cells. In control cells, inhibiting P450 2E1 through chemical or siRNA approaches suppressed ROS accumulation, ER membrane lipid peroxidation and the resultant cell death after ER stress. However, it had little effect in BI1-overexpressing cells. In addition, BI1 knock down also increased ROS accumulation and expression of P450 2E1. In a reconstituted phospholipid membrane containing purified BI1, NPR and P450 2E1, BI1 dose-dependently decreased the production of ROS. BI1 bound to NPR with higher affinity than P450 2E1. Furthermore, BI1 overexpression reduced the interaction of NPR and P450 2E1, and decreased the catalytic activity of P450 2E1, suggesting that the flow of electrons from NPR to P450 2E1 can be modulated by BI1. In summary, BI1 reduces the accumulation of ROS and the resultant cell death through regulating P450 2E1.

Regulation of Cytochrome P450 2E1 under Hypertonic Environment through TonEBP in Human Hepatocytes

Whereas the liver as well as the other organs are continually exposed to the change of osmotic status, it has never been investigated whether activities and gene expressions of drug-metabolizing enzymes, including cytochromes P450, are dependent on osmotic change in the liver. In the present study, we determined that CYP2E1 is induced under hypertonic environments at a transcriptional level in human primary hepatocytes, as assessed by cDNA microarray and real time-reverse transcription-polymerase chain reaction analyses. Both a protein level and the catalytic activity of CYP2E1 were consistently increased in response to hypertonic conditions. In promoter-reporter assay, it was demonstrated that -586 to -566 in the CYP2E1 5'-flanking region was necessary for 2E1 promoter activation by hypertonic stimulation. It is noteworthy that tonicity-response element (TonE) consensus sequence was found at -578 to -568 in human CYP2E1 5'-flanking region, and electrophoretic mobility shift assay demonstrated the interaction of TonE binding protein (TonEBP) with TonE motif of CYP2E1 promoter. Furthermore, cotransfection of a CYP2E1 promoter construct with wild-type TonEBP expression vector enhanced promoter activity under both isotonic and hypertonic conditions, whereas dominant-negative TonEBP suppressed an induction of CYP2E1 promoter activity. These results indicate that the level of CYP2E1 is induced by hypertonic condition via TonEBP transactivation. The present study suggests that osmotic status may influence individual responses to the substrate of CYP2E1.

The regulation of cytochrome P450 2E1 during LPS-induced inflammation in the rat

It is well known that inflammatory and infectious conditions differentially regulate cytochrome P450 (P450)-mediated drug metabolism in the liver. We have previously outlined a potential pathway for the downregulation in hepatic cytochrome P450 following LPS-mediated inflammation in the CNS (Abdulla, D., Goralski, K.B., Garcia Del Busto Cano, E., Renton, K.W., 2005. The signal transduction pathways involved in hepatic cytochrome P450 regulation in the rat during an LPS-induced model of CNS inflammation. Drug Metab. Dispos). The purpose of this study was to outline the effects of LPS-induced peripheral and central nervous system inflammation on hepatic cytochrome P450 2E1 (CYP2E1) in vivo, an enzyme that plays an important role in various physiological and pathological states. We report an increase in hepatic mRNA expression of CYP2E1 that occurred as early as 2-3 h following either the intraperitoneal (i.p.) injection of 5 mg/kg LPS or i.c.v. administration of 25 mug of LPS. This increase in CYP2E1 mRNA expression was sustained for 24 h. In sharp contrast to the increase in hepatic CYP2E1 mRNA, we observed a significant reduction in the catalytic activity of this enzyme 24 h following either the i.c.v. or i.p. administration of LPS. Cycloheximide or actinomycin-D did not change the LPS-mediated downregulation in hepatic CYP2E1 catalytic activity. Our results support the idea that LPS acts at two different levels to regulate hepatic CYP2E1: a transcriptional level to increase CYP2E1 mRNA expression and a post-transcriptional level to regulate CYP2E1 protein and activity.

Regulation of Cytochrome P450 2E1 by Heat Shock Protein 90-Dependent Stabilization and CHIP-Dependent Proteasomal Degradation

Alcohol-inducible cytochrome P450 2E1 (CYP2E1) has the most rapid turnover of any member of this large family of membrane-bound oxygenases, and its degradation rate is altered profoundly by various substrates, such as ethanol and CCl(4). CYP2E1 is degraded by the ubiquitin-proteasome pathway, and because the hsp90/hsp70-based chaperone machinery is often involved in maintaining the balance between protein integrity and degradation by this pathway, we have asked whether CYP2E1 is regulated by the chaperone machinery. We show here that treatment of transformed human skin fibroblasts stably expressing CYP2E1 with the hsp90 inhibitor radicicol results in CYP2E1 degradation that is inhibited by the proteasome inhibitor lactacystin. Immunoadsorption of hsp90 from cytosol of HEK cells expressing the truncated CYP2E1(Delta3-29) yields coadsorption of CYP2E1(Delta3-29). Cotransfection of HEK cells with both the truncated CYP2E1 and the hsp70-dependent E3 ubiquitin ligase CHIP results in CYP2E1(Delta3-29) degradation, and CYP2E1(Delta3-29) co-immunoadsorbs with myc-CHIP from cytosol of cotransfected cells. Purified, bacterially expressed CYP2E1(Delta3-29) is ubiquitylated in a CHIP-dependent manner when it is incubated with a purified system containing the E1 ubiquitin activating enzyme, E2, and CHIP. CYP2E1 is the first P450 shown to be an hsp90 "client" protein that can be ubiquitylated by the hsp70-dependent E3 ubiquitin ligase CHIP. Our observations lead to a general model of how substrates, such as ethanol, can regulate the interaction of CYP2E1 with the chaperones hsp90 and hsp70 to profoundly alter enzyme turnover.

Protective effect of Lycium chinense fruit on carbon tetrachloride-induced hepatotoxicity

In the present study, we investigated the protective effect of Lycium chinense Miller (Solanaceae) fruit (LFE) against CCl 4-induced hepatotoxicity and the mechanism underlying these protective effects in rats. The pretreatment of LFE has shown to possess a significant protective effect by lowering the serum aspartate and alanine aminotransferase (AST and ALT) and alkaline phosphatase (ALP). This hepatoprotective action was confirmed by histological observation. In addition, pretreatment of LFE prevented the elevation of hepatic malondialdehyde (MDA) formation and the depletion of reduced glutathione (GSH) content and catalase activity in the liver of CCl 4-injected rats. The LFE also displayed hydroxide radical scavenging activity in a dose-dependent manner (IC 50 = 83.6 μg/ml), as assayed by electron spin resonance (ESR) spin-trapping technique. The expression level of cytochrome P450 2E1 (CYP2E1) mRNA and protein, as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analysis, was significantly decreased in the liver of LFE-pretreated rats when compared with that in the liver of control group. Based on these results, it was suggested that the hepatoprotective effects of the LFE might be related to antioxidative activity and expressional regulation of CYP2E1.

Differential effects of dietary fatty acids on the regulation of CYP2E1 and protein kinase C in human hepatoma HepG2 cells.

We investigated the effects of different fatty acids (FAs) or with different degrees of unsaturation on cytochrome P450 2E1 (CYP2E1) induction and protein kinase C (PKC) activity in human hepatoma HepG2 cells. As the degree of unsaturation increased, the cell survival rate decreased for FAs with 18 carbons, but linolenic acid (LNA) or docosahexaenoic acid (DHA) groups were similar even through they have different degrees of unsaturation. Treatment with palmitic acid (PA), oleic acid (OA), linoleic acid (LA), LNA, and DHA resulted in respective cellular FA concentrations of C16:0 (43.1%), C18:1 (18.5%), C18:2 (7.4%), LNA (2.85%), and C22:6 (3.13%), which was highest for the FA that was used as the treatment, indicating that their incorporation within the cell is directly proportional to treatment. After 2 hours of cultivation, the lipid peroxide (LPO) in the DHA group increased 600% compared with control, and was much higher than in the groups treated with the other FAs, with LNA > LA > OA > PA. CYP2E1 induction increased with greater effect as the degree of unsaturation of OA, LA, and DHA increased. PA did not affect PKC activity, but DHA treatment increased PKC activity the most. The effects of LNA and LA were similar, but less than that of DHA, and that of OA was lower still, indicating that activity of PKC is proportional to the degree of unsaturation, and not the configuration of the FA. Increased plasma membrane concentrations of n-3 FA, such as DHA, might exert regulatory effects on PKC by increasing membrane fluidity, causing changes in CYP2E1, elevating levels of LPO, or producing oxidative stress.

A novel lipopolysaccharide-modulated Jun binding repressor in intron 2 of CYP2E1.

Cytochrome P450 2E1 (CYP2E1) exhibits a pronounced oxidase activity that may mediate apoptotic injury in glial cells as well as hepatocytes. Strict regulation of CYP2E1 and it's activity is therefore thought to be crucial. We have studied CYP2E1 transcriptional regulation in primary cortical glial cells and have identified a novel repressor element at +1452/+1460 in intron 2 of the rat CYP2E1 gene. The element very potently repressed CYP2E1 and SV40 promoters and consisted of the non-palindromic core sequence 5'-TTCCACTCA-3'. Jun proteins were found to interact with the site. The protein complexes were also found to contain an as yet unidentified protein of approximately 60 kDa, probably with DNA binding properties similar to G-box binding factors found in, e.g. Arabidopsis thaliana. Stimulation with lipopolysaccharide, or overexpression of the mitogen-activated protein kinase kinase kinase, MEKK-1, further deepened the repression in primary cortical glial cells. It is suggested that this novel Jun binding repressor helps to control basal expression levels of CYP2E1, and modulates the response to inflammatory factors. Future in vivo experiments will, however, be required for a full appreciation of the role of this repressor in the complex regulation of CYP2E1 during inflammatory conditions.

CYP2E1 Degradation by in Vitro Reconstituted Systems: Role of the Molecular Chaperone hsp90

One major mode of regulation of cytochrome P450 2E1 (CYP2E1) is at the posttranscriptional level, since many low-molecular-weight compounds stabilize the enzyme against proteolysis by the proteasome complex. In an in vitro system containing human liver microsomes, degradation of CYP2E1 in the microsomes required addition of the human liver cytosol fraction in a reaction sensitive to inhibitors of the proteasome complex. It is not clear how CYP2E1 in the microsomal membrane becomes accessible to the cytosolic proteasome. Since molecular chaperones play a role in protein folding and degradation, the possible role of heat shock proteins in CYP2E1 degradation by this reconstituted system was evaluated. Degradation of CYP2E1 required ATP; ATP-γS, a nonhydrolyzable analogue of ATP, did not catalyze CYP2E1 degradation by the cytosol fraction, indicating that ATP hydrolysis is required. Geldanamycin, a specific inhibitor of hsp90, inhibited the degradation of microsomal CYP2E1 by the cytosol fraction. Control experiments indicated that geldanamycin was not a substrate/ligand of CYP2E1 nor did it prevent microsomal lipid peroxidation, a process which increases CYP2E1 turnover. Inhibition by geldanamycin was prevented by molybdate. Both of these compounds have been shown to promote alterations in hsp90 structure and to modulate hsp90–protein interactions. The proteasome activity in the cytosol, as assayed by the cleavage of a fluorogenic peptide, was enhanced when ATP was added and inhibited by 30–40% by geldanamycin, effects that are similar, although less pronounced, to the degradation of CYP2E1 by the cytosol. Purified 20S proteasome could catalyze degradation of CYP2E1; however, in an assay using equal peptidase activity, the cytosol fraction was much more effective than the 20S proteasome in promoting CYP2E1 degradation. Immunodepletion of hsp90 from the cytosol resulted in prevention of the degradation of CYP2E1, a reaction that was reversed by the addition of pure hsp90 to this cytosol. These results suggest that in addition to the proteasome, the cytosol fraction contains other factors that modulate the efficiency of CYP2E1 degradation. The sensitivity to geldanamycin and molybdate and the immunodepletion experiments suggest that hsp90 is one of these factors that interact with CYP2E1 and/or with the proteasome to promote the degradation of this microsomal P450.

Expression of cytochrome P4502E1 in human liver: assessment by mRNA, genotype and phenotype.

Cytochrome P4502E1 (CYP2E1) is constitutively expressed in human liver and is responsible for the metabolic bioactivation of a wide variety of xenobiotics, including a number of protoxins and procarcinogens. CYP2E1 expression is regulated at several levels including pre-transcriptional, transcriptional and post-transcriptional levels, and any variation in enzyme concentration and hence activity may represent increased risk of toxicity or carcinogenicity. We have investigated variability in the levels of CYP2E1 mRNA, protein and functional activity in a human liver bank, and attempted to relate these parameters to the RsaI restriction fragment length polymorphism in the 5'-flanking region. Variation in CYP2E1 mRNA (18-fold) was greater than the variation seen in CYP2E1 protein (twofold) and functional activity (fourfold) determined using two probe substrates, chlorzoxazone and p-nitrophenol. Although protein and functional activity showed a significant correlation (r = 0.93 and r = 0.83 for chlorzoxazone and p-nitrophenol, respectively), there was no correlation between any of these parameters and mRNA levels. Also, the variation in CYP2E1 activity could not be directly accounted for by the RsaI polymorphism in our samples. In conclusion, our results are consistent with a complex regulation of CYP2E1 and the fact that it is highly conserved in the human population. The absence of a relationship between the RsaI polymorphism and CYP2E1 activity is consistent with other studies performed in Caucasians, but does not exclude an effect of this polymorphism on inducibility of CYP2E1.

PHOSPHORYLATION OF CYTOCHROME P4502E1 (CYP2E1) BY CALMODULIN DEPENDENT PROTEIN KINASE PROTEIN KINASE C AND cAMP DEPENDENT PROTEIN KINASE

Phosphorylation of pure cytochrome P4502E1 (CYP2E1) was achieved in vitro using Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), protein kinase C (PKC) and cAMP-dependent protein kinase (PKA). The stoichiometry and time-course of phosphorylation were determined. CaM kinase II was the most efficient enzyme capable of catalyzing this phosphorylation reaction: the maximum incorporation of 32PO4 was 0.8 mol/mol CYP2E1 in 20 min. PKA phosphorylated a maximum of 0.7 mol of 32PO4/mol of cytochrome within 60 min. The phosphorylation by PKC reached a maximum of 0.19 mol of 32PO4/mol of cytochrome and this occurred within a few minutes of incubation. Limited digestion by S. aureus V8 protease (SAP) of CYP2E1, which had been phosphorylated by either PKA and PKC, yielded a single major phosphopeptide with an M(r) of approximately 18,000. Limited digestion of CYP2E1, that had been phosphorylated by CaM kinase II, yielded phosphorylated polypeptides with M(r) of approximately 18,000 and 15,000. These results raise the possibility that these three kinases may be involved in the regulation of CYP2E1.